Enzymatic degumming of edible oils comprising a relatively high amount of non-hydratable phosphorus content:
It is known to use phospholipase for enzymatic degumming of a water degummed edible oil (U.S. Pat. No. 5,264,367, Metallgesellschaft, Rohm), to reduce the phosphorous content of said water degummed edible oil.
However, this process may still be improved, especially to perform enzymatic degumming of edible oils comprising a high amount of non-hydratable phosphorus (NHP) and/or relatively high amounts of mucilage.
Consequently, an object of the invention is to provide a method for reducing the content of phosphorus containing components of such oils, wherein said method comprises use of a phospholipase.
A Phospholipase of the invention
Phospholipids, such as lecithin or phosphatidylcholine, consist of glycerol esterified with two fatty acids in an outer (sn-1) and the middle (sn-2) positions and esterified with phosphoric acid in the third position; the phosphoric acid, in turn, may be esterified to an amino-alcohol. Phospholipases are enzymes which participate in the hydrolysis of phospholipids. Several types of phospholipase activity can be distinguished, including phospholipases A.sub.1 (PLA.sub.1) and A.sub.2 (PLA.sub.2) which hydrolyze one fatty acyl group (in the sn-1 and sn-2 position, respectively) to form lysophospholipid; and lysophospholipase (or phospholipase B (PLB)) which can hydrolyze the remaining fatty acyl group in lysophospholipid.
This invention relates i.a. to a filamentous fungal phospholipase that has the ability to hydrolyze one and/or both fatty acyl groups in a phospholipid (i.e. exhibiting PLA and/or PLB activity).
Previolusly characterized fungal PLA and/or PLB enzymes:
Numerous references describe characterization of fungal phospholipases. In order to make it easier to get an overview of the prior art status, the references have been grouped in two sections.
Section one deals with references describing the identification of fungal phospholipases presently not believed to be related to the fungal phospholipase of the present invention. These references are mainly included in order to summarize the state of the art within the field of characterization of fungal phospholipases.
Section two deals with references describing characterization of fungal phospholipases believed to be of some relevance to the fungal phospholipases of the present invention.
Section one
Enzymes with phospholipase A and/or B activity have been found in various fungal sources, including Penicillium notatum (also known as P. chrysogenum; N. Kawasaki, J. Biochem., 77, 1233-44, 1975; N. Masuda et al., Eur. J. Biochem., 202, 783-787, 1991), P. cyclopium (Process Biochemistry 30(5): 393-401 (1995)), Saccharomyces cerevisiae (M. Ichimasa et al., Agric. Biol. Chem., 49 (4), 1083-89, 1985; F. Paultauf et al., J. Biol. Chem., 269, 19725-30, 1994), Torulaspora delbrueckii (old name Saccharomyces rosei; Y. Kuwabara, Agric. Biol. Chem., 52 (10), 2451-58, 1988; FEMS, Microbiol. Letters, 124, 29-34), Schizosaccharomyces pombe (H. Oishi et al., Biosci. Biotech. Biochem., 60 (7), 1087-92, 1996), Aspergillus niger (Technical Bulletin, G-zyme.TM. G999, Enzyme Bio-Systems Ltd.; Process Biochemistry 30(5): 393-401 (1995)) and Corticium centrifugum (S. Uehara et al., Agric. Biol. Chem., 43 (3), 517-525, 1979).
Section two:
EP 575133 A2 describes isolation and characterization of a fungal phoshoslipase A1 obtained from Aspergillus and the use thereof for industrial applications.
No sequence information (neither DNA nor amino acid) is disclosed in the application nor is any strategy or suggestion for cloning any of the Aspergillus phospholipase discussed or indicated in the application.
Tsung-Che et al. (Phytopathological notes 58:1437-38 (1968)) briefly describes characterization of a phospholipase from Fusarium solani.
EP 130,064 describes an isolated fraction of a fermentation broth exhibiting lipase activity obtained from the strain Fusarium oxysporum, DSM 2672. Furthermore, the use thereof in detergent compositions is disclosed. However, EP 130,064 does not describe this fraction as exhibiting any phospholipase activity.
WO 96/13579 describes a lipase obtained from the strain Fusarium culmorum, CBS 513.94 including its N-terminal sequence.
However, WO 96/13579 does not describe any enzyme exhibiting phospholipase activity.
A cDNA sequence encoding a lipase from Fusarium heterosporum is described (Cloning and nucleotide sequence of cDNA encoding a lipase from Fusarium heterosporum, J. Biochem. 116, 536-540, 1994.). This sequence is presently believed to be the most related DNA sequence compared to a cloned DNA sequence of the invention (See section "Comparison with prior art" (vide infra). However, this reference does not describe any enzyme exhibiting phospholipase activity.
A cDNA sequence encoding a phospholipase B from Penicillum notatum is described (Eur. J. Biochem 202:783-787, 1991). However this cloned DNA sequence has very limited homology to a DNA sequence of the invention (See section "Comparison with prior art" (vide infra)
Industrial application of phospholipases:
A number of uses of phospholipases are known in the art, such as to use phospholipase in, e.g. enzymatic degumming of a water degummed oil (U.S. Pat. No. 5,264,367, Metallgesellschaft, Rohm); treatment of starch hydrolysate (particularly from wheat starch) to improve the filterability (EP 219,269, CPC International); as an additive to bread dough to improve the elasticity of the bread (U.S. Pat. No. 4,567,046, Kyowa Hakko); and for preparation of lysolecithin with special emulsifying properties.
Presently, the phospholipase Lecitase.RTM. (Novo Nordisk A/S) is used commercially such as for degumming of oils. Lecitase.RTM. is a mammalian enzyme obtained from porcine pancreas.
It is well known that it is possible to produce fungal enzymes recombinantly at industrially economically acceptable yields, especially from filamentous fungi.
Consequently, it is an object of this invention to provide an improved phospholipase e.g. for use in the processes described above.
Further, it is an object of the present invention to describe processes and methods for recombinant production at industrially acceptable yields of a phospholipase obtained from a filamentous fungus.